The discovery of the green fluorescent protein (GFP) in 1962 by Osamu Shimomura and coworkers (1) was a seminal event that spawned subsequent revolution in genetically encoded fluorescent proteins. Since then, GFP has remained the archetypal fluorescent protein and has received the greatest attention and widest application (2). GFP and other fluorescent proteins are now solidly foundational tools in cell biology and numerous biotechnological applications including molecular probes (3). Past and current work on GFP and other fluorescent proteins may be conceptually divided into the pursuit of general improvements as well as specific application development (4, 5). Notable general advancements include the development of brighter fluorescence, higher photostability, alternative colors, and defined oligomerization state (6-11). More specific applications are enabled by specialized variants that are sensitive to pH, oxygen tension, cation concentrations, or other environmental conditions; recently, this has been further extended to photoswitching/photoactivation behavior that has been leveraged for super-resolution imaging applications (12-15).
Novel variants of existing fluorescent proteins as well as new fluorescent proteins continue to be an active area of research (2). Typically, control of both spectral and biochemical characteristics has been sought. Spectrally, this includes increasing or decreasing brightness, photostability, and quantum yield and modulation of excitation and emission peaks and curves. Biochemically, control of protein oligomerization, stability, folding kinetics, and other interactions (either up or down for all of these) enable different usage scenarios as well (6, 9, 16-20). At the core, spectral characteristics, primarily overall brightness during imaging applications (a complex function of excitation quantum cross section, quantum yield for fluorescence, Stokes shift, and specific imaging system components) are still deeply useful when improved. This has driven the extensive in vitro mutagenesis and evolution of GFP as well as the search for other naturally fluorescent proteins (4, 13, 17, 18, 20-22). Simplistically, this pursuit of brightness still seems reasonable because small molecule (i.e., non-genetically encodable) fluorophores continue to boast higher overall brightness than fluorescent proteins (ex. fluorescein is still brighter than all GFP variants (10). Therefore, variants of GFP and other fluorescent proteins that exhibit improved overall brightness are still valuable additions to the fluorescent protein toolbox and, if biochemical characteristics are preserved, are immediately available for upgrading most existing applications.
The key biochemical feature of GFP that enables the vast majority of applications is its ability to spontaneously fold as a single domain, resulting in a (generally) facile, nontoxic, stable fusion partner for a variety of proteins (11). Fusion to a protein of interest enables studies of protein localization, interactions, and stability (3, 23-25). Fusion to antigen binding proteins, notably antibodies, enables a large array of in vivo and in vitro affinity tagging, purification, identification, and visualization applications (7, 9, 13, 24, 26-29). Recently, identification of single domain antibodies, or nobodies, that bind to GFP and modulate its fluorescence has resulted in several additional novel tools for further application development (30-33). Like GFP itself, nanobodies are ideal fusion partners that fold spontaneously into a single stable domain (34-36). Thus, these GFP binding nanobodies have been used for intentional association of proteins (one fused to GFP and one fused to the nanobody) in vivo, resulting in an enhanced or reduced fluorescence readout (33). Furthermore, chromobodies couple a nanobody with arbitrary specificity to any fluorophore for detection, enabling in vivo as well as in vitro fluorescent molecular probe or localization reagents (31, 37-40). Despite several reported exceptions (24, 27, 41), however, one major drawback for antibody-fluorescent protein fusions remains the general production of these reagents, as the disulfide bonds often required in nanobodies and other antibodies typically require oxidizing conditions that preclude proper folding of GFP (39) and thereby require modified host strains (39, 42-44) or tolerating low periplasmic yields (43, 45-47).
Recent results have demonstrated that one GFP variant, termed sfGFP (48), is capable of folding properly in the bacterial periplasm (49-51), where most variants (notably wtGFP and EGFP) are very dim or nonfluorescent. Furthermore, sfGFP has been shown to enhance the proper folding of protein domains fused to it (48). Therefore, in addition to expanding the use of GFP variants to the study of the bacterial periplasm and eukaryotic endoplasmic reticulum compartments, sfGFP could have an additional benefit in improving cytoplasmic expression of antibody domains.